The uptake of iron into mammalian cells involves the binding of transferrin, the serum iron transport protein, to the tranferin receptor, followed by the internalization of the receptor-transferrin complex. Iron accumulation is essential for cell division. Rapidly proliferating cells have higher iron requirements than non-dividing cells and the receptor has been identified as a proliferation specific marker. The transferrin receptor contains three asparagine-linked oligosaccharides, which are a mixture of "high mannose" and "complex" structures and at least one O-linked (serine/threonine) oligosaccharide. Prior studies using tunicamycin-treated human cells have demonstrated that the unglycosylated from of the transferrin receptor is not functional. Thus, the goals of this proposal are to test the hypothesis that glycosylation is vital for the correct folding and transport of the transferrin receptor to the cell surface and in the binding or transferrin. Site-directed mutagenesis will be used to test the contribution of each of the asparagine-linked glycosylation sites to the folding and transport of the transferrin receptor. Combinations of single, double and triple mutations will be prepared and tested mouse cell lines. Evidence suggests that processing of oligosaccharides to complex forms enhances receptor function and the role of this processing will be examined in glycosylation deficient Chinese Hamster Ovary cell mutants. Such mutants will also be sued to investigate the role of O-linked glycosylation in receptor function. Transferrin receptor oligosaccharides have not been directly characterized and they will be analyzed with respect to structure (high mannose/complex) present on each of the asparagine-linked sites in the native transferrin receptor as well as the receptor expressed in murine and CHO cells following transfection. These analyses will be performed via carbohydrate and protein chemical techniques, including - reverse phase HPLC, peptides sequencing and analysis of oligosaccharides on lectin affinity and molecular sieve columns.